Mechanisms of RPA, Recombinases, and Mediators in Homologous Recombination

项目总结 同源重组(HR)是维持基因组稳定性的关键，其功能是 消除DNA双链断裂和染色体损伤。人力资源调节剂的突变决定了 许多癌症和遗传性疾病的病理进展。当双链DNA出现时，HR就会启动 Break被核溶解切除以产生单链DNA(SsDNA)突出物，这些突出物很容易被包被 并受复制蛋白A(RPA)保护。乳腺癌2型易感性(BRCA2)蛋白 作用是去除和重塑RPA并促进RAD51重组酶的结合，然后催化 链交换和驱动重组。我们的长期目标是从机械上理解 这些HR介体之间的单链DNA移交的时间序列以及这些介体如何竞争 获得单链DNA及其对癌症等疾病的总体贡献。RPA由多个不同的 DNA结合域(DBD)，与单链DNA具有高亲和力。固有的DNA结合动力学(结合， 单个DBD的分离和重塑)被假定为决定RPA的时间、地点和方式 功能。几十年来，RPA的四个DBD被分为高亲和力(DBDS-A和B)和低亲和力 亲和力(DBDS-C&D)基于分离的DBDS与单链DNA结合亲和力的实验测量 已经形成了DNA复制、修复和重组中RPA机制的模型。使用非规范 氨基酸，我们开发了一种基于荧光的方法来捕获单个DBDS在 全长蛋白质的上下文。与经典模型相反，我们发现高亲和力的DBD是 动态的，并被RPA的三聚化核心所击败。代替这些令人兴奋的RPA发现 机制，我们在这里建议揭示BRCA2介体如何影响RPA-DBDS的动力学 在HR过程中促进RAD51丝的形成。我们将确定DNA的背景和类型 人力资源中遇到的结构会影响RPA-DBD动态[目标1]。我们将调查以下问题的重要性 RPA中的一个调控热点，将调节RPA与BRCA2-DSS1复合体之间的相互作用 [目标2]。最后，使用BRCA2、RAD51和RAD52的荧光版本，我们将建立 在HR期间从RPA到RAD51的ssDNA移交[目标3]。

PROJECT SUMMARY Homologous recombination (HR) is critical for the maintenance of genomic stability, and functions to eliminate DNA double-strand breaks and chromosomal lesions. Mutations in HR mediators dictate the pathological progression of many cancers and hereditary disorders. HR is initiated when a double-stranded DNA break is nucleolytically resected to generate single-stranded DNA (ssDNA) overhangs, which are readily coated and protected by Replication Protein A (RPA). The Breast Cancer Type 2 Susceptibility (BRCA2) protein functions to remove and remodel RPA and promote binding of the RAD51 recombinase, which then catalyzes strand exchange and drives recombination. Our long-term goals are to gain a mechanistic understanding of the temporal sequence of ssDNA handoff between these HR mediators and how these mediators compete for access to ssDNA and its overall contribution to diseases such as cancer. RPA is composed of multiple distinct DNA binding domains (DBDs) and binds with high affinity to ssDNA. The intrinsic DNA binding dynamics (binding, dissociation and remodeling) of individual DBDs are hypothesized to dictate when, where, and how RPA functions. For several decades the four DBDs of RPA have be classified as high affinity (DBDs-A & B) and low affinity (DBDs-C & D) based on experimental measurements of ssDNA binding affinity of isolated DBDs and have shaped models for RPA mechanism in DNA replication, repair and recombination. Using non-canonical amino acids, we developed a fluorescence-based method to capture the dynamics of individual DBDs in the context of the full-length protein. In contrary to classical models, we uncovered that the high-affinity DBDs are dynamic and are outcompeted by the trimerization core of RPA. In lieu of these exciting discoveries of RPA mechanism, we here propose to uncover how the BRCA2 mediator influences the dynamics of RPA-DBDs to promote the formation of the RAD51 filament during HR. We will establish how the context and type of DNA structures that are encountered in HR affect RPA-DBD dynamics [Aim 1]. We will investigate the importance of a regulatory hotspot in RPA that would modulate the interaction between RPA and the BRCA2-DSS1 complex [Aim 2]. Finally, using fluorescent versions of BRCA2, RAD51 and RAD52, we will establish the mechanism of ssDNA handoff from RPA to RAD51 during HR [Aim 3].